Non Alcoholic Steatohepatitis (NASH) is generally recognized as a serious progression of the more common Non-alcoholic fatty liver disease (NAFLD). NAFLD is a precursor state to NASH, because only with NASH is there a pattern of inflammation that leads to fibrosis and eventually cirrhosis. Ordinarily, fewer than 20% of patients with NAFLD progress to NASH, although those who progress account for the majority of the severe complications discussed in this application (1). NASH has become the third-leading cause of liver transplants in the U.S., with anywhere between 2% and 5% of the general population currently affected. Since 2001, liver transplants attributed to NASH have increased by an alarming 1,000%. By 2025, it's estimated that more than 25 million Americans will have hepatic steatosis with at least detectable fibrosis. For perspective, Hepatitis C affects just 3.2 million.
The main culprit for this rapid increase in NASH is none other than the rising incidence of Metabolic Syndrome (MetS) diseases, which are all linked to progression of underlying insulin resistance.
Type 2 diabetes mellitus (T2D), obesity and dyslipidemia are the principal MetS component factors associated with NAFLD, which is now considered the hepatic expression of MetS. Several studies have dealt with the relationship of NAFLD and MetS, the risk of liver disease associated with the classical features of MetS, and the importance of insulin resistance as the precursor condition of different MetS constituent diseases. In the past 10 years alone, the rate of obesity has doubled in adults and tripled in children. Not only does obesity contribute to the inherent cause of NASH, but it also increases the risk of T2D and high blood cholesterol, which can further complicate the health of a patient with NASH. Resolution of insulin resistance will resolve NAFLD, because triglycerides are lowered and the fatty deposits are primarily comprised of these lipids.
Both NAFLD and NASH are closely associated with insulin resistance, and insulin resistance is thereby linked to progression to hepatocellular carcinoma (2, 3).
The epidemics of obesity and diabetes of Western countries is expected to produce a significant increase of MetS associated liver disease in the next years. We still need to clarify the mechanism(s) responsible for liver disease progression from pure fatty liver, to steatohepatitis and to cirrhosis, and the reason(s) why only a few NAFLD cases actually progress to terminal liver failure while others (the majority) will have a cardiovascular outcome before the liver is end-stage. Prevention and intervention programs based on lifestyle are therefore mandatory to reduce the burden of metabolic liver disease.(4)
Nonalcoholic fatty liver disease is now recognized as the most common liver disease in the United States, with a prevalence of approximately 5% in the general population and up to 25% to 75% in patients with obesity and T2D. Nonalcoholic fatty liver disease is a clinicopathologic syndrome with a wide spectrum of histologic abnormalities and clinical outcomes. Hepatic steatosis has a benign clinical course, and it appears almost completely reversible if there is weight loss and a reduction in insulin resistance. In contrast, nonalcoholic steatohepatitis (NASH) may progress to cirrhosis and liver-related death in 25% and 10% of patients, respectively. Cases occur most commonly in obese, middle-aged women with diabetes. However, NASH may also occur in children and normal-weight men with normal glucose and lipid metabolism. The pathophysiology involves two steps. The first is insulin resistance caused by dietary excess and hepatic overload, recognized as steatosis. The second is oxidative stress, which produces lipid peroxidation and activates inflammatory cytokines resulting in NASH. Liver biopsy provides prognostic information and identifies NASH patients who may benefit from therapy.(5)
RYGB surgery definitively resolves insulin resistance, normalizes triglycerides, and thus completely resolves NAFLD. RYGB surgery also lowers inflammation and mitigates hepatocellular damage from inflammation, leading to resolution of elevated liver enzymes (ALT, AST). Studies in our laboratories have shown a resolution of systemic inflammation with RYGB(6), and studies by others have shown that RYGB not only prevents progression of fibrosis, but reverses at least some of the biopsy proven fibrosis in patients with NASH (7-12). In these RYGB patients, the first biopsy was at the time of surgery, when there was elevated ALT and AST as well as demonstrable hepatic fibrosis. When these patients had normalization of their elevated liver enzymes, in most cases they were considered to have resolved hepatic inflammation, and repeating the biopsy was not considered medically necessary. Thus while essentially all RYGB cases have normalization of insulin resistance, ALT, AST and Triglycerides, there are relatively small numbers of RYGB cases in the literature with biopsy proven resolution of fibrosis.
Imaging studies also show resolution of both hepatic steatosis (NAFLD) and fibrosis (NASH) after RYGB (13-15). These non-invasive methods are recognized as supporting evidence for approval in NASH, and it is possible that in the future, regulatory agencies may consider these methods as equivalent to biopsy.
Mathematical Models of NASH and NAFLD have not been developed, although there is the potential for such models to assist with diagnosis, risk stratification, and defining the effects of the various treatments. It would be a major advance in this field to have an integrated model of the disease based on biomarkers and non-invasive tests. Such a model must be predictive of the biopsy results however, in order for it to be considered useful for diagnosis, patient stratification and for monitoring treatment response.
On the basis of the laboratory biomarkers and the RYGB results of others to date, Brake™ is a promising approach to both NAFLD and NASH. As the first oral mimetic of RYGB surgery, Brake™ has thus far normalized insulin resistance, triglycerides, ALT, AST, and hyperglycemia. Except for RYGB surgery, no therapy has yet resolved either of these conditions, and RYGB itself is not indicated as a treatment for NASH, although patients with NASH conditions do have RYGB surgery if they qualify for the surgery overall.
The current standard for definitive diagnosis of NAFLD or NASH is a biopsy of the liver, which is an expensive and invasive procedure. Because this procedure is done very infrequently, and at great cost to the patients and their caregivers, we lack a readily available method to assess the effects of drug treatments for NAFLD or NASH. Single laboratory tests such a measurements of liver enzymes ALT or AST do not predict the total response of the liver, so the idea of non-invasively defining drug response and other useful effects has not advanced in medical practice nor in clinical research. It is a novel idea of the present invention to present a means of predicting changes in liver biopsy using simultaneous modeling of groups of biomarkers and laboratory tests. Efforts to date in this regard focus primarily on single parameter predictions, and have yielded some success, but overall the applicability of single parameters to predict biopsy endpoints in new patients has met with only marginal success.
Both NAFLD and NASH are Associated with Death from CV Causes
A complex interaction among metabolic factors, adipose tissue lipolysis, oxidative stress, and insulin resistance results in a deleterious process that may link nonalcoholic fatty liver disease (NAFLD) with severe cardiovascular (CV) outcomes such as myocardial infarction and stroke. Patients with NAFLD are at higher risk of atherosclerosis, new onset of CV events, and overall mortality. The strong association between NAFLD and CV disease should affect clinical practice, with screening and surveillance of patients with NAFLD(16)
Bril and colleagues studied patients with NAFLD in order to determine the contribution of the severity of steatohepatitis to atherogenic dyslipidemia. The study was conducted at a university hospital. Patients were recruited from outpatient clinics or from the general population (n=188). Patients had measurement of hepatic triglyceride content by magnetic resonance spectroscopy, histology (liver biopsy), metabolic profile by means of an oral glucose tolerance test, and lipoprotein analyses were performed. Outcomes measured included standard lipids, lipoprotein sub-fraction analysis (apolipoprotein B/A1 levels, low-density lipoprotein (LDL) particle size/phenotype, and LDL/high-density lipoprotein sub-fractions), and insulin resistance. Patients with NAFLD had severe insulin resistance, especially at the level of the adipose tissue, when compared with patients without NAFLD. Despite small differences in triglycerides and high-density lipoprotein-cholesterol, patients with NAFLD had a significantly higher plasma apolipoprotein B to apolipoprotein A1 ratio (0.66+/−0.02 vs 0.58+/−0.02, P=0.01) and smaller LDL particle size (216.2+/−0.7 vs 219.4+/−1.1 A, P=0.01). Of note, these differences between patients with/without NAFLD were independent of the presence of obesity. Severity of steatohepatitis did not significantly influence the lipoprotein profile. Worse atherogenic dyslipidemia was best predicted by the degree of liver fat accumulation and adipose tissue and systemic insulin resistance. Bril concluded that NAFLD was associated with a worse atherogenic lipoprotein profile, regardless of similar body mass index and other clinical parameters. They speculate that this lipoprotein profile is driven mostly by liver fat content and insulin resistance and appears not to be worsened by obesity or the severity of liver disease (17)
Non-alcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular related death, particularly in those with hepatic fibrosis. Long and colleagues determined the prevalence of predicted fibrosis based on non-invasive fibrosis markers and the association of hepatic fibrosis with cardiovascular risk factors in a Cross-sectional study of 575 Framingham Heart Study participants with NAFLD based on computed tomography. They determined the prevalence of predicted fibrosis based on the aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, AST to platelet ratio index (APRI), the Fibrosis-4 score (FIB4), and the NAFLD Fibrosis Score (NFS). Using multivariable logistic regression models, they examined the association between low, indeterminate, or high risk for fibrosis according to the NFS and various cardiometabolic risk factors. The predicted risk of fibrosis was 12%, 4%, 5%, and 32% for the NFS, FIB4, APRI, and AST/ALT ratio, respectively. In multivariable models, participants with a high risk for advanced fibrosis by the NFS had a wider pulse pressure (adjusted mean difference=6.87 mm Hg; p=0.0002) and increased odds of hypertension (OR 2.92; p=0.007) compared to those with low risk of fibrosis. There were no statistically significant differences between other cardiovascular risk factors for those with a high versus low risk of fibrosis. The AST/ALT ratio, APRI, and NFS give widely disparate predictions of liver fibrosis. Participants with a high risk for fibrosis based on NFS had wider pulse pressure and increased odds of hypertension. Whether modifying these risk factors impacts cardiovascular endpoints in NAFLD patients remains unknown.(18).
While epidemiological studies have attempted to measure age acceleration and increased mortality rates in those with concurrent metabolic syndrome and chronic viral infections such as HCV and HIV, such measurements are made difficult by the myriad co-factors. For instance, metabolic syndrome associated disorders such as diabetes, HCV infection, and medication adherence are important factors of HIV infection that are also suspected to significantly affect mortality rates. While most previous studies have not attempted to control for these factors, the study by Gross and colleagues has focused specifically on well-characterized subjects. Their estimate of HIV age advancement was 4.9 years, calculated from a quantitative analysis of the methylome.(19). They state that further work will be needed to understand if the observed epigenetic age advancement is generalizable to broader slices of the HIV+ population (i.e., patients with complex co-morbidities such as drug use or additional viral infections). This study is based on the same metabolic syndrome mediated epigenetic model of biological aging as many others, including recent reports associating epigenetic aging increases of 6.6 years with Down's Syndrome(20), traumatic stress (21), and even all-cause mortality (22). Recent studies have identified biomarkers of chronological age based on DNA methylation levels. It is not yet known whether DNA methylation age captures aspects of biological age. Marioni and colleagues tested whether differences between people's chronological ages and estimated ages, DNA methylation age, predict all-cause mortality in later life. The difference between DNA methylation age and chronological age was calculated in four longitudinal cohorts of older people. Meta-analysis of proportional hazards models from the four cohorts was used to determine the association between chronological age and mortality. A 5-year higher chronological age is associated with a 21% higher mortality risk, adjusting for age and sex. After further adjustments for childhood IQ, education, social class, hypertension, diabetes, cardiovascular disease, and APOE e4 status, there is a 16% increased mortality risk for those with a 5-year higher Chronological age. Metabolic syndrome progression is intrinsic to the aging process because it pre-stages the onset of diseases which shorten lifespan, such as diabetes, NASH, Alzheimer's, Hypertension, Myocardial infarction, renal failure and CHF. Accordingly, it may be predicted that a roll back in the rate of progression of MetS, such as can be shown by RYGB or treatment with an ileal brake hormone releasing composition, could lengthen life expectancy by many years. As MetS can now be staged with risks in mind using calculated FS index and CV index, it follows that use of ileal brake hormone releasing substances in patients with MetS may extend the time before they develop these life-shortening conditions.
Inflammation measured as high sensitivity C-reactive protein (hsCRP) also manifests as a complication of MetS, and in a landmark study, weight loss (−11.23 kg; 95% confidence interval, −11.54 to −10.92; P<0.001) reduced not only hsCRP, but also low- and high-density lipoprotein cholesterol, triglycerides, and blood pressure. During the 26-week weight maintenance period in the intention-to-treat analysis, the further decrease of hsCRP blood levels was −0.46 mg/L greater (95% confidence interval, −0.79 to −0.13) in the groups assigned to low-glycemic-index diets than in those on high-glycemic-index diets (P<0.001). Groups on low-protein diets achieved a −0.25 mg/L greater reduction in hsCRP (95% confidence interval, −0.59 to −0.17) than those on high-protein diets (P<0.001), whereas lipid profiles and blood pressure were not differently affected(23). RYGB also lowers many biomarkers of inflammation(6), and it would be expected that the preferred embodiments of an ileal brake hormone releasing composition would also lower inflammation and thus lower the risks of complications of inflammation such as NASH in the liver.
The inflammation that converts NAFLD to NASH can arise from insulin resistance, abdominal obesity, or these chronic viral infections. Among the infectious causes of NASH, one must consider the rising incidence of Hepatitis C, which is also rapidly increasing worldwide. Hepatitis C infects 2-3% of the world's population, over 180 million persons, and is a cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma(24). The standard of care in the recent past was pegylated interferon plus ribavirin (pegIFN/Riba) combination therapy, although this therapy is both expensive and poorly tolerated. Treatment efficacy is approximately 50%. Telaprevir and boceprevir, two direct acting antiviral (DAA) protease inhibitors, have recently been approved for clinical use in the US(25). Addition of either of these new agents has the potential to improve sustained virological response in hepatitis C to 65-75%. However, the addition of a DAA to the current standard of care introduces the risk of side effects, including anemia and rash, and failure to achieve Sustained Viral Response (SVR) may pose an increased risk of accumulation protease inhibitor-resistant viral strains that may carry over resistance problems to future treatments. None the less, newer agents such as sofosbuvir (Sovaldi) and these in combination treatments have steadily increased the percentage of patients who achieve SVR in Hepatitis C treatments. Shorter courses of treatment to achieve SVR, as well as fewer side effects have both been advantages of these newer DAA antivirals
None of these current or future treatments appear to provide any benefit to the patient beyond suppression of the virus. Specifically, the liver is typically not healed even when viral counts are very low. The hepatic damage may cease when there is SVR, or inflammation and associated fibrosis may even progress slowly in the presence of a small number of residual viral particles which presumably persist inside hepatocytes. Hepatic steatohepatitis, the primary accompanying condition of most patients with hepatitis C, continues and may progress even with complete viral suppression, and it is now time to propose the controversial position that NAFLD and NASH must be managed in lock step with Hepatitis C, even with the newest DAA antivirals and associated treatments. Likewise, it is possible that interferons were prematurely abandoned as combinations with DAAs, as there are recent papers that propose that Hepatocellular carcinoma incidence remains high even after SVR(26-28). On this basis, it is the goal of the present invention to lower the risk of Hepatocellular carcinoma, given that one beneficial aspect of the ileal brake hormone releasing substance treatment has been to lower Alpha Fetoprotein concentration.
Regardless of cause, NAFLD is a common diagnosis in populations as a whole, often as frequent as 25%(29). There is no FDA approved drug therapy for either NAFLD or NASH as of this writing in early 2017 (30), and most experts rely on lifestyle counseling alone. Of great concern, NASH is a histologic feature in approximately two thirds of liver biopsies of patients with chronic hepatitis C. Until recently, this common finding was not carefully documented, and there were no large longitudinal studies describing the progression of steatosis in chronic hepatitis C or even hepatitis B. In 2009, Lok and colleagues examined changes in steatosis on serial biopsies among chronic hepatitis C patients participating in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) trial(31). All 1050 patients in this trial had advanced fibrosis at baseline biopsy (NASH criteria) and were documented not to have had a sustained virological response to pegIFN/Riba. Most (94%) of these patients had genotype 1 infection. At least one protocol follow-up biopsy was read on 892 patients, and 699 had the last biopsy performed 3.5 years after randomization. Hepatic damage was well advanced at enrollment, as 39% had cirrhosis and 61% had bridging fibrosis; 18%, 41%, 31%, and 10% had steatosis scores of 0, 1, 2, and 3 or 4, respectively. The mean steatosis score decreased in the follow-up biopsies in both the pegIFN/Riba-treated patients and controls with no effect of treatment assignment (P=0.66). A decrease in steatosis score by > or =1 point was observed in 30% of patients and was associated with both progression to cirrhosis and continued presence of cirrhosis (P=0.02). Compared to patients without a decrease in steatosis, those with a decrease in steatosis had worse metabolic parameters at enrollment, and were more likely to have a decrease in alcohol intake, improvement in metabolic parameters, and worsening liver disease (cirrhosis, esophageal varices, and deterioration in liver function). Lok and colleagues(31) concluded that hepatic steatosis recedes during progression from advanced fibrosis to cirrhosis. However, there was no available means to produce a decline in either NAFLD or NASH in most patients with Hepatitis C, which then became the primary motivation to discover a means of treating hepatic steatosis as an integral part of treatment of hepatitis C patients.
In a further definitive examination of the role of hepatic steatosis on the course of hepatitis C therapy, Briceno and colleagues (2009) examined livers that were to be transplanted into patients with hepatitis C that had already destroyed the original liver (32). The aim of this study was to determine the influence of donor graft steatosis on overall outcome, viral recurrence, and fibrosis progression in orthotopic liver transplantation for hepatitis C virus cirrhosis. One hundred twenty patients who underwent OLT for HCV cirrhosis between 1995 and 2005 were included in the study. Donor steatosis was categorized as absent (0%-10%; n=40), mild (10%-30%; n=32), moderate (30%-60%; n=29), or severe (>60%; n=19). A Cox multivariate analysis for marginal donor variables and a Model for End-Stage Liver Disease index were performed. Fibrosis evolution was analyzed in liver biopsies (fibrosis <2 or > or =2) 3, 6, and 12 months post-OLT and in the late post-OLT period. Fifty-six grafts were lost (46%). The survival of the grafts was inversely proportional to donor liver steatosis: 82%, 72%, and 72% at 1, 2, and 3 years post-OLT in the absence of steatosis; 73%, 63%, and 58% with mild steatosis; 74%, 62%, and 43% with moderate steatosis; and 62%, 49%, and 42% with severe steatosis (P=0.012). HCV recurrence was earlier and more frequent in recipients with steatosis >30% (46% versus 32% at 3 months, P=0.017; 58% versus 43% at 6 months, P=0.020; 70% versus 56% at 12 months, P=0.058; and 95% versus 69% at 3 years post-OLT, P=0.0001).(32). Graft survival was lower in alcoholic liver disease recipients versus HCV recipients when steatosis was >30% at 3, 6, and 12 months post-OLT (P=0.042) but not when steatosis was <30% (P=0.53). A higher fibrosis score was obtained 3 months post-OLT (P=0.033), 6 months post-OLT (P=0.306), 12 months post-OLT (P=0.035), and in the late post-OLT period (P=0.009). The authors concluded that the degree of hepatic steatosis in the new liver greatly influences the recurrence of hepatitis C and its progression in the new liver. In fact, Steatosis affects the success of treatment the second time. Hepatitis C recurrence was more frequent and earlier in recipients of moderately and severely steatotic livers. Fibrosis evolution is more rapid and severe when graft steatosis is >30% (32). As pointed out by Lok as well, there is a need to manage the hepatic steatosis in order to optimize the outcome of antiviral therapy for hepatitis C.
Testino and colleagues (2009) examined the influence of improvement in MetS (typically associated with hepatic steatosis) biomarkers on the response of patients with hepatitis C to pegIFN/Riba(33). They examined baseline biomarkers such as Body Mass Index (BMI), cholesterol, triglycerides (TGs) and hepatic percentage of steatosis in the response to therapy with pegIFN/Riba in patients with recurrent hepatitis C (genotype 1). In this study, 30 consecutive prospectively followed patients diagnosed with recurrent hepatitis C were considered candidates for antiviral therapy. The observed distribution of BMI, cholesterol, TGs and steatosis were confirmed to be normally distributed by the one-sample Kolmogorov-Smirnov Goodness of fit test procedure. Comparison of BMI, cholesterol, TGs and steatosis between non responders (NR), sustained virological responders (SVR) and sustained biochemical responders (SBR) groups were analyzed by ANOVA with a post hoc Bonferroni test and correlation between variables was tested by Pearson test. The multivariate analysis was performed to estimate the chance of response on basis of the above-mentioned variables. In patients with abnormal results in at least two out of four steatosis-associated variables, the chance of no-response was 40 times higher than that of SBR and 96 times than that of SVR(33). On the basis of these epidemiological studies, they argued that diet and exercise therapy should improve BMI, liver histology and, therefore, the response to pegIFN/Riba(33). Indeed this study provides further justification for concomitant use of a treatment for hepatic steatosis in conjunction with a treatment for the hepatitis C virus itself. This unmet need is addressed by the present invention, an ileal brake hormone releasing composition that acts in a similar manner to RYGB surgery on MetS components in human patients.
There is also evidence that management of hepatic steatosis in patients with hepatitis C would be of value in the prevention of hepatocellular carcinoma (HCC). For example, Pekow and colleagues (2007) (34) retrospectively identified 94 consecutive patients with hepatitis C cirrhosis who underwent liver transplantation from 1992 to 2005 and had pathology available for review. Of these, 32 had evidence of HCC, and 62 had no HCC on explant histology. All explant specimens were then graded for steatosis by a single, blinded pathologist. Next, hepatic steatosis, age, sex, BMI, HCV RNA, HCV genotype, Model for End-Stage Liver Disease (MELD) score, chronic alcohol use, and diabetes were examined in univariate and multivariate analyses for association with HCC. In total, 69% of patients in the HCC group and 50% of patients in the control group had evidence of hepatic steatosis (1+) on histology. Odds ratios for the development of HCC for each grade of steatosis compared with grade 0 were as follows: grade 1 (1.61 [0.6-4.3]), grade 2 (3.68 [1.1-12.8]), and grade 3 or 4 (8.02 [0.6-108.3]) (P=0.03 for the trend). In univariate analyses, there was a significant association between increasing steatosis grade (P=0.03), older age (56 years versus 49 years; P<0.02), higher ALT aspartate aminotransferase (122.5 U/L vs. 91.5 U/L; P=0.005), higher AST alanine aminotransferase (95.8 U/L vs. 57.2 U/L; P=0.002), higher alpha-fetoprotein (113.5 ng/mL vs. 17.8 ng/mL; P<0.001), lower median HCV RNA (239,000 IU/mL vs. 496,500 IU/mL; P=0.02), higher biologic MELD score (21.8 vs. 20.3; P=0.03), and risk of HCC. In multivariate analysis, age (P=0.02), alpha-fetoprotein (P=0.007), and hepatic steatosis (P=0.045) were significantly associated with HCC(34). These authors concluded that in patients with Hepatitis C-related cirrhosis, the presence of hepatic steatosis is independently associated with the development of hepatocellular carcinoma(34). Clearly if the NAFLD and NASH could be reversed by a companion treatment to the anti-viral agent, there is plausible evidence that HCC might be prevented or at least there would be fewer cases that progress to this deadly complication of the combined problem of hepatitis C and NASH. The surprising finding of recent studies, is that the DAAs do not lower the risk of HCC and in fact may even increase it in settings where the HCC process has already started on a sub-clinical basis.(26-28)